The proliferation and differentiation of cardiac fibroblasts (CFs) are critical for the heart's adaptation to pathological stresses (Heling et al., Circ. Res. 86:846-53, 2000). Specifically, CF activity immediately after myocardial infarction (Cameron et al., Endocrinol. 141:4690-7, 2000) and during cardiac remodeling (Katz, J. Cell. Mol. Med. 7:1-10, 2003; Brown et al., Annu. Rev. Pharmacol. Toxicol. 45:657-87, 2005) leads to myocardial fibrosis and the elaboration of collagen and extracellular matrix, the degree of which largely determines the outcome of clinical heart failure (Brown et al., Annu. Rev. Pharmacol. Toxicol. 45:657-87, 2005; Bax et al., Circulation. 110:1118-22, 2004).
Atrial (ANP) and brain (BNP) natriuretic peptide (NPs) are produced in the heart and potently inhibit CFs through their ability to bind and activate the ubiquitous NP receptor, known as natriuretic peptide receptor A or NPRA (Silberbach and Roberts, Cell. Signal. 13:221-31, 2001). Thus, the NP-NPRA system serves as an endogenous defense against maladaptive cardiac hypertrophy (Molkentin, J. Clin. Invest. 111: 1275-7, 2003; Silberbach et al., J. Biol. Chem. 274:24858-64, 1999). However, in the clinical setting of heart failure, receptor resistance limits these beneficial downstream effects (Fan et al., Mol. Pharmacol. 67:174-83, 2005; Tsutamoto et al., Circ. 87:70-5, 1993; Nakamura et al., Am. Heart J. 135:414-20, 1998; Kuhn et al., Cardiovasc. Res. 64:308-14, 2004). Monogenetic mouse models mimic the condition of heart failure-induced NPRA resistance, in which either cardiac-restricted NPRA deletion (Holtwick et al., J. Clin. Invest. 111: 1399-407, 2003) or expression of a dominant-negative NPRA mutant produces load-independent cardiac hypertrophy and fibrosis (Patel et al., Am. J. Physiol. Heart Circ. Physiol. 289:H777-84, 2005). However, in vivo, desensitization of the NPRA receptor is not likely due to such mutations. Therefore, models of the in vivo situation are needed, for example to identify agents that are likely to restore function to NPRA in vivo.
NPRA exists as a homodimer prior to ligand binding. Phosphorylated NPRA is active, and decreased phosphorylation causes receptor desensitization. However, a specific NPRA kinase has not been identified (Potter and Hunter, J. Biol. Chem. 276:6057-60, 2001). NP binding to the extracellular domain is thought to induce a conformational change in the receptor that results in the juxtaposition of the C-terminal guanylyl cyclase domains of the respective NPRA monomers, leading to the generation of 5′-cyclic-guanosine-monophosphate (cGMP). cGMP serves as a second messenger that activates cGMP-dependent protein kinase I (PKG). PKG mediates many NP downstream effects such as cardiac hypertrophy and fibrosis (Silberbach and Roberts, Cell. Signal. 13:221-31, 2001) and promotes cardiomyocyte survival (Kato et al., J. Clin. Invest. 115:2716-30, 2005).
While searching for distal PKG binding partners, an association between PKG and a C-terminal fragment of NPRA (NPRA(820-1061)) was identified (Airhart et al., J. Biol. Chem. 278:38693-8, 2003). PKG I is a cytosolic serine-threonine kinase that is expressed in a variety of tissues, including the heart and peripheral vasculature. Small quantities of membrane-associated PKG I in NPRA over-expressing HEK 293 cells (HEK-NPRA cells), which increased significantly following NP treatment.
Although administration of recombinant NPs has been recently approved by the FDA, use of such compounds is limited due to NPRA resistance, which always occurs in heart failure. In addition, the use of such recombinant NPs may have deleterious long-term effects that lead to kidney failure and increased hospital mortality. Therefore, there is a need to identify additional compounds that can be used to treat cardiovascular diseases, such as heart failure.